A Pattern Recognition Receptor C-type Lectin-S6 (CTL-S6) is Involved in the Immune Response in the Silkworm (Lepidoptera: Bombycidae).

Insect innate immunity is initiated by the special recognition and binding of the foreign pathogens, which is accomplished by the pattern recognition receptors (PRRs). As an important type of PRRs, C-type lectins (CTLs) play various roles in insect innate immunity, including pathogen recognition, stimulation of prophenoloxidase, regulation of cellular immunity and so on. In this study, we have cloned the full-length cDNA of a CTL gene named CTL-S6 from the silkworm, Bombyx mori. The open reading frame (ORF) of B. mori CTL-S6 encodes 378 amino acids, which contain a secretion signal peptide. The mRNA of CTL-S6 exhibited the highest transcriptional level in the midgut. Its transcriptional level increased dramatically in fat body and hemocytes upon Escherichia coli or Micrococcus luteus challenge. Purified recombinant CTL-S6 could bind to bacterial cell wall components, including peptidoglycan (PGN, from Bacillus subtilis) and lipopolysaccharide (LPS, from E. coli 0111:B4), and recombinant CTL-S6 was involved in the encapsulation and melanization of hemocytes. Furthermore, the addition of recombinant CTL-S6 to the hemolymph of silkworm resulted in a significant increase in phenoloxidase activity. Overall, our results indicated that B. mori CTL-S6 may serve as a PRR for the recognition of foreign pathogens, prophenoloxidase pathway stimulation and involvement in the innate immunity.

Shen D ，Tong M ，Guo J ，Mei X ，Xia D ，Qiu Z ，Zhao Q ... -  《JOURNAL OF INSECT SCIENCE》

A single-CRD C-type lectin is important for bacterial clearance in the silkworm.

C-type lectins (CTLs) depend on the carbohydrate-recognition domain (CRD) to recognize carbohydrates by a Ca(2+)-dependent mechanism. In animals, CTLs play critical roles in pathogen recognition, activation of the complement system and signaling pathways. Immulectins (Dual-CRD CTLs) in lepidopteran are involved in recognizing pathogens. However, little is known about the immune-related functions of insect single-CRD CTLs. Here, we reported the characterization of C-type lectin-S3 (CTL-S3), a single-CRD CTL from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S3 gene is 672 bp, which encodes a putative protein of 223 amino acids. CTL-S3 gene was expressed in a variety of tissues. Levels of CTL-S3 mRNA in fertilized eggs and whole larvae were elevated upon bacterial challenges. CTL-S3 was secreted to larval hemolymph. The recombinant protein (rCTL-S3) binds to bacterial cell wall components and bacteria. CTL-S3 inhibited the growth of Bacillus subtilis and caused agglutination of Staphylococcus aureus. More importantly, CTL-S3 facilitated the rapid clearance of Escherichia coli and Staphylococcus aureus from the body cavity of larvae. Taken together, our results suggested that CTL-S3 may function as an opsonin in larval hemolymph to enhance the clearance of pathogens.

Zhan MY ，Shahzad T ，Yang PJ ，Liu S ，Yu XQ ，Rao XJ ... -  《-》

Identification of C-type lectin-domain proteins (CTLDPs) in silkworm Bombyx mori.

C-type lectins (CTLs) represent a large family of proteins that can bind carbohydrate moieties normally in a calcium-dependent manner. CTLs play important roles in mediating cell adhesion and the recognition of pathogens in the immune system. In the present study, we have identified 23 CTL genes in domestic silkworm Bombyx mori. CTL-domain proteins (CTLDPs) are classified into three groups based on the number of carbohydrate-recognition domains (CRDs) and the domain architectures. These include twelve CTL-S (Single-CRD), six immulectins (Dual-CRD) and five CTL-X (CRD with other domains). We studied their phylogenetic features, analyzed the conserved residues, predicted tertiary structures, and examined the tissue expression profile and immune inducibility. Through bioinformatics analysis, we have putatively identified ten secretory and two cytoplasmic CTL-S; four secretory and two cytoplasmic immulectins; one secretory, one cytoplasmic and three transmembrane forms of CTL-X. Most B. mori CTLDPs form monophyletic groups with orthologs from Lepidoptera, Diptera, Coleoptera and Hymenoptera species. Immulectins of B. mori and Manduca sexta evolved from common ancestor genes perhaps due to gene duplication events of CTL-S ancestor genes. Homology modeling revealed that the overall structures of B. mori CTL domains are analogous to those of humans with a variable loop region. We examined the expression profile of CTLDP genes in naïve and immune-stimulated tissues. The expression and induction of CTLDP genes were related to the tissues and microorganisms. Together, our gene identification, sequence comparison, phylogenetic analysis, homology modeling and expression analysis laid a good foundation for the further studies of B. mori CTLDPs and comparative genomics.

Rao XJ ，Shahzad T ，Liu S ，Wu P ，He YT ，Sun WJ ，Fan XY ，Yang YF ，Shi Q ，Yu XQ ... -  《-》

Peptidoglycan recognition protein-S1 (PGRP-S1) from Diaphania pyloalis (Walker) is involved in the agglutination and prophenoloxidase activation pathway.

Recognition of invading foreign exogenous pathogen is the first step to initiate the innate immune response of insects, which accomplished by the pattern recognition receptors (PRRs). Peptidoglycan recognition proteins (PGRPs) serve as an important type of PRRs, which activate immune response by detecting peptidoglycan of microbial cell wall. In this study, we have cloned the full-length cDNA of PGRP gene called PGRP-S1 from the Diaphania pyloalis (Walker). The open reading frame (ORF) of D. pyloalis PGRP-S1 encodes 211 amino acids which containing a secretion signal peptide and a canonical PGRP domain. Multisequence alignment revealed that PGRP-S1 possess the amino acid residues responsible for zinc binding and amidase activity. D. pyloalis PGRP-S1 exhibited the highest transcript level in fat body and followed in head. The mRNA concentration dramatically increased after an injection of Escherichia coli or Micrococcus luteus. Purified recombinant PGRP-S1 exhibit binding ability to peptidoglycans from Staphylococcus aureus or Bacillus subtilis and cause intensive agglutination of E. coli, M. luteus or S. aureus in the presence of zinc ions. Furthermore, phenoloxidase activity significantly increased when the plasma from larvae was incubated with recombinant PGPR-S1 and peptidoglycans from B. subtilis or M. luteus simultaneously. These results implied that PGRP-S1 was a member involving the prophenoloxidase activation pathway. Overall, our results indicated that D. pyloalis PGRP-S1 serve as a PRR to participate in the recognition of foreign pathogen and prophenoloxidase pathway stimulation.

Shen D ，Mei X ，Guo J ，Tong M ，Xia D ，Qiu Z ，Zhao Q ... -  《-》

Molecular cloning and analysis of a C-type lectin from silkworm Bombyx mori.

C-type lectins (CTLs) play a variety of roles in plants and animals. They are involved in animal development, pathogen recognition, and the activation of immune responses. CTLs carry one or more non-catalytic carbohydrate-recognition domains (CRDs) to bind specific carbohydrates reversibly. Here, we report the molecular cloning and functional analysis of a single-CRD CTL, named C-type lectin-S2 (BmCTL-S2) from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S2 is 666 bp, which encodes a putative protein of 221 amino acids. BmCTL-S2 is expressed in a variety of immune-related tissues, including hemocytes and fat body among others. BmCTL-S2 mRNA level in the midgut and the fat body was significantly increased by bacterial challenges. The recombinant protein (rBmCTL-S2) bound different bacterial cell wall components and bacterial cells. rBmCTL-S2 also inhibited the growth of Bacillus subtilis and Staphylococcus aureus. Taken together, we infer that BmCTL-S2 is a pattern-recognition receptor with antibacterial activities.

Shahzad T ，Zhan MY ，Yang PJ ，Yu XQ ，Rao XJ ... -  《-》